Transmembrane helix-helix interactions: comparative simulations of the glycophorin a dimer

The glycophorin helix dimer is a paradigm for the exploration of helix-helix interactions in integral membrane proteins. Two NMR structures of the dimer are known, one in a detergent micelle and one in a lipid bilayer. Multiple (4 x 50 ns) molecular dynamics simulations starting from each of the two NMR structures, with each structure in either a dodecyl phosphocholine (DPC) micelle or a dimyristoyl phosphatidylcholine (DMPC) bilayer, have been used to explore the conformational dynamics of the helix dimer. Analysis of the helix-helix interaction, mediated by the GxxxG sequence motif, suggests convergence of the simulations to a common model. This is closer to the NMR structure determined in a bilayer than to micelle structure. The stable dimer interface in the final simulation model is characterized by (i) Gly/Gly packing and (ii) Thr/Thr interhelix H-bonds. These results demonstrate the ability of extended molecular dynamics simulations in a lipid bilayer environment to refine membrane protein structures or models derived from experimental data obtained in protein/detergent micelles.     

Modeling Transmembrane Domain Dimers/Trimers of Plexin Receptors: Implications for Mechanisms of Signal Transmission across the Membrane

Single-pass transmembrane (TM) receptors transmit signals across lipid bilayers by helix association or by configurational changes within preformed dimers. The structure determination for such TM regions is challenging and has mostly been accomplished by NMR spectroscopy. Recently, the computational prediction of TM dimer structures is becoming recognized for providing models, including alternate conformational states, which are important for receptor regulation. Here we pursued a strategy to predict helix oligomers that is based on packing considerations (using the PREDDIMER webserver) and is followed by a refinement of structures, utilizing microsecond all-atom molecular dynamics simulations. We applied this method to plexin TM receptors, a family of 9 human proteins, involved in the regulation of cell guidance and motility. The predicted models show that, overall, the preferences identified by PREDDIMER are preserved in the unrestrained simulations and that TM structures are likely to be diverse across the plexin family. Plexin-B1 and –B3 TM helices are regular and tend to associate, whereas plexin-A1, -A2, –A3, -A4, -C1 and –D1 contain sequence elements, such as poly-Glycine or aromatic residues that distort helix conformation and association. Plexin-B2 does not form stable dimers due to the presence of TM prolines. No experimental structural information on the TM region is available for these proteins, except for plexin-C1 dimeric and plexin-B1 – trimeric structures inferred from X-ray crystal structures of the intracellular regions. Plexin-B1 TM trimers utilize Ser and Thr sidechains for interhelical contacts. We also modeled the juxta-membrane (JM) region of plexin-C1 and plexin-B1 and show that it synergizes with the TM structures. The structure and dynamics of the JM region and TM-JM junction provide determinants for the distance and distribution of the intracellular domains, and for their binding partners relative to the membrane. The structures suggest experimental tests and will be useful for the interpretation of future studies.     

Multiple binding modes for palmitate to barley lipid transfer protein facilitated by the presence of proline 12

Molecular dynamics simulations have been used to characterise the binding of the fatty acid ligand palmitate in the barley lipid transfer protein 1 (LTP) internal cavity. Two different palmitate binding modes (1 and 2), with similar protein–ligand interaction energies, have been identified using a variety of simulation strategies. These strategies include applying experimental protein–ligand atom–atom distance restraints during the simulation, or protonating the palmitate ligand, or using the vacuum GROMOS 54B7 force‐field parameter set for the ligand during the initial stages of the simulations. In both the binding modes identified the palmitate carboxylate head group hydrogen bonds with main chain amide groups in helix A, residues 4 to 19, of the protein. In binding mode 1 the hydrogen bonds are to Lys 11, Cys 13, and Leu 14 and in binding mode 2 to Thr 15, Tyr 16, Val 17, Ser 24 and also to the OH of Thr 15. In both cases palmitate binding exploits irregularity of the intrahelical hydrogen‐bonding pattern in helix A of barley LTP due to the presence of Pro 12. Simulations of two variants of barley LTP, namely the single mutant Pro12Val and the double mutant Pro12Val Pro70Val, show that Pro 12 is required for persistent palmitate binding in the LTP cavity. Overall, the work identifies key MD simulation approaches for characterizing the details of protein–ligand interactions in complexes where NMR data provide insufficient restraints.     

Structural and thermodynamic insight into the process of “weak” dimerization of the ErbB4 transmembrane domain by solution NMR

Specific helix–helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure–function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651–678)₂ form a right-handed parallel dimer through the N-terminal double GG4-like motif A⁶⁵⁵GxxGG⁶⁶⁰ in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer–dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true (“ideal”) solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix–helix interactions, which may be critical for membrane protein activity.     

Spatial structure and pH-dependent conformational diversity of dimeric transmembrane domain of the receptor tyrosine kinase EphA1

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.     

Talin‐driven inside‐out activation mechanism of platelet αIIbβ3 integrin probed by multimicrosecond,all‐atom molecular dynamics simulations

Platelet aggregation is the consequence of the binding of extracellular bivalent ligands such as fibrinogen and von Willebrand factor to the high affinity, active state of integrin αIIbβ3. This state is achieved through a so‐called “inside‐out” mechanism characterized by the membrane‐assisted formation of a complex between the F2 and F3 subdomains of intracellular protein talin and the integrin β3 tail. Here, we present the results of multi‐microsecond, all‐atom molecular dynamics simulations carried on the complete transmembrane (TM) and C‐terminal (CT) domains of αIIbβ3 integrin in an explicit lipid‐water environment, and in the presence or absence of the talin‐1 F2 and F3 subdomains. These large‐scale simulations provide unprecedented molecular‐level insights into the talin‐driven inside‐out activation of αIIbβ3 integrin. Specifically, they suggest a preferred conformation of the complete αIIbβ3 TM/CT domains in a lipid‐water environment, and testable hypotheses of key intermolecular interactions between αIIbβ3 integrin and the F2/F3 domains of talin‐1. Notably, not only do these simulations give support to a stable left‐handed reverse turn conformation of the αIIb juxtamembrane motif rather than a helical turn, but they raise the question as to whether TM helix separation is required for talin‐driven integrin activation. Proteins 2014; 82:3231–3240. © 2014 Wiley Periodicals, Inc.     

Pore Structure and Synergy in Antimicrobial Peptides of the Magainin Family

Magainin 2 and PGLa are among the best-studied cationic antimicrobial peptides. They bind preferentially to negatively charged membranes and apparently cause their disruption by the formation of transmembrane pores, whose detailed structure is still unclear. Here we report the results of 5–9 μs all-atom molecular dynamics simulations starting from tetrameric transmembrane helical bundles of these two peptides, as well as their stoichiometric mixture, and the analog MG-H2 in DMPC or 3:1 DMPC/DMPG membranes. The simulations produce pore structures that appear converged, although some effect of the starting peptide arrangement (parallel vs. antiparallel) is still observed on this timescale. The peptides remain mostly helical and adopt tilted orientations. The calculated tilt angles for PGLa are in excellent agreement with recent solid state NMR experiments. The antiparallel dimer structure in the magainin 2 simulations resembles previously determined NMR and crystal structures. More transmembrane orientations and a larger and more ordered pore are seen in the 1:1 heterotetramer with an antiparallel helix arrangement. Insights into the mechanism of synergy between these two peptides are obtained via implicit solvent modeling of homo- and heterodimers and analysis of interactions in the atomistic simulations. This analysis suggests stronger pairwise interactions in the heterodimer than in the two homodimers.     

Structural and thermodynamic insight into the process of "weak" dimerization of the ErbB4 transmembrane domain by solution NMR

Specific helix-helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure-function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651-678)(2) form a right-handed parallel dimer through the N-terminal double GG4-like motif A(655)GxxGG(660) in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer-dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true ("ideal") solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix-helix interactions, which may be critical for membrane protein activity.     

Modulation of glycophorin A transmembrane helix interactions by lipid bilayers: molecular dynamics calculations

Starting from the glycophorin A dimer structure determined by NMR, we performed simulations of both dimer and monomer forms in explicit lipid bilayers with constant normal pressure, lateral area, and temperature using the CHARMM potential. Analysis of the trajectories in four different lipids reveals how lipid chain length and saturation modulate the structural and energetic properties of transmembrane helices. Helix tilt, helix-helix crossing angle, and helix accessible volume depend on lipid type in a manner consistent with hydrophobic matching concepts: the most relevant lipid property appears to be the bilayer thickness. Although the net helix-helix interaction enthalpy is strongly attractive, analysis of residue-residue interactions reveals significant unfavorable electrostatic repulsion between interfacial glycine residues previously shown to be critical for dimerization. Peptide volume is nearly conserved upon dimerization in all lipid types, indicating that the monomeric helices pack equally well with lipid as dimer helices do with one another. Enthalpy calculations indicate that the helix-environment interaction energy is lower in the dimer than in the monomer form, when solvated by unsaturated lipids. In all lipid environments there is a marked preference for lipids to interact with peptide predominantly through one rather than both acyl chains. Although our trajectories are not long enough to allow a full thermodynamic treatment, these results demonstrate that molecular dynamics simulations are a powerful method for investigating the protein-protein, protein-lipid, and lipid-lipid interactions that determine the structure, stability and dynamics of transmembrane alpha-helices in membranes.     

Ligand recognition by A‐class Eph receptors: crystal structures of the EphA2 ligand‐binding domain and the EphA2/ephrin‐A1 complex

Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B‐class receptors. Here, we present the crystal structures of an A‐class complex between EphA2 and ephrin‐A1 and of unbound EphA2. Although these structures are similar overall to their B‐class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A‐class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a ‘lock‐and‐key’‐type binding mechanism, in contrast to the ‘induced fit’ mechanism defining the B‐class molecules. This model is supported by structure‐based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell‐based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2‐specific homotypic cell adhesion interactions.     

Molecular dynamics simulation of the C-terminal sterile alpha-motif domain of human p73alpha: evidence of a dynamical relationship between helices 3 and 5

We used molecular dynamics simulation to evaluate the association properties of C-terminal sterile alpha-motif (SAM) domain of human p73alpha. To test the dimerization propensity of this structure we carried out four simulations: EphB2 X-ray dimer, p73 modeled dimer, p73 NMR monomer, and p73 modeled monomer with an elongated helix 5. The results show a direct interaction between helix 5 and helix 3 since a conformational collapse of helix 3 is observed when dimer contact and/or an elongation of helix 5 is introduced by modeling in p73 SAM domain. On the basis of these results we suggest that the recognition properties of the SAM domains may be modulated by the conformational state of helix 5.     

Thermodynamics of the pseudo‐knot in helix 18 of 16S ribosomal RNA

A fragment of E. coli 16S rRNA formed by nucleotides 500 to 545 is termed helix 18. Nucleotides 505‐507 and 524‐526 form a pseudo‐knot and its distortion affects ribosome function. Helix 18 isolated from the ribosome context is thus an interesting fragment to investigate the structural properties and folding of RNA with pseudo‐knots. With all‐atom molecular dynamics simulations, spectroscopic and gel electrophoresis experiments, we investigated thermodynamics of helix 18, with a focus on its pseudo‐knot. In solution studies at ambient conditions we observed dimerization of helix 18. We proposed that the loop, containing nucleotides forming the pseudo‐knot, interacts with another monomer of helix 18. The native dimer is difficult to break but introducing mutations in the pseudo‐knot indeed assured a monomeric form of helix 18. Molecular dynamics simulations at 310 K confirmed the stability of the pseudo‐knot but at elevated temperatures this pseudo‐knot was the first part of helix 18 to lose the hydrogen bond pattern. To further determine helix 18 stability, we analyzed the interactions of helix 18 with short oligomers complementary to a nucleotide stretch containing the pseudo‐knot. The formation of higher‐order structures by helix 18 impacts hybridization efficiency of peptide nucleic acid and 2'‐O methyl RNA oligomers.     

Energetics of ErbB1 Transmembrane Domain Dimerization in Lipid Bilayers

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as −2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.     

Specificity of helix packing in transmembrane dimer of the cell death factor BNIP3: a molecular modeling study

BNIP3 is a mitochondrial 19-kDa proapoptotic protein, a member of the Bcl-2 family. It has a single COOH-terminal transmembrane (TM) alpha-helical domain, which is required for membrane targeting, proapoptotic activity, hetero- and homo-dimerization in membrane. The role and the molecular details of association of TM helices of BNIP3 are yet to be established. Here, we present a molecular modeling study of helix interactions in its membrane domain. The approach combines Monte Carlo conformational search in an implicit hydrophobic slab followed by molecular dynamics simulations in a hydrated full-atom lipid bilayer. The former technique was used for exhaustive sampling of the peptides' conformational space and for generation of putative "native-like" structures of the dimer. The latter ones were taken as realistic starting points to assess stability and dynamic behavior of the complex in explicit lipid-water surrounding. As a result, several groups of tightly packed right-handed structures of the dimer were proposed. They have almost similar helix-helix interface, which includes the motif A(176)xxxG(180)xxxG(184) and agrees well with previous mutagenesis data and preliminary NMR analysis. Molecular dynamics simulations of these structures reveal perfect adaptation of most of them to heterogeneous membrane environment. A remarkable feature of the predicted dimeric structures is the occurrence of a cluster of H-bonded histidine 173 and serines 168 and 172 on the helix interface, near the N-terminus. Because of specific polar interactions between the monomers, this part of the dimer has no such dense packing as the C-terminal one, thus allowing penetration of water from the extramembrane side into the membrane interior. We propose that the ionization state of His(173) can mediate structural and dynamic properties of the dimer. This, in turn, may be related to pH-dependent proapoptotic activity of BNIP3, which is triggering on by acidosis appearing under hypoxic conditions.     

High resolution solution structure of apo calcyclin and structural variations in the S100 family of calcium-binding proteins

The three-dimensional solution structure of apo rabbit lung calcyclin has been refined to high resolution through the use of heteronuclear NMR spectroscopy and 13C,15N- enriched protein. Upon completing the assignment of virtually all of the 15N, 13C and 1H NMR resonances, the solution structure was determined from a combination of 2814 NOE- derived distance constraints, and 272 torsion angle constraints derived from scalar couplings. A large number of critical inter- subunit NOEs (386) were identified from 13C- select,13C-filtered NOESY experiments, providing a highly accurate dimer interface. The combination of distance geometry and restrained molecular dynamics calculations yielded structures with excellent agreement with the experimental data and high precision (rmsd from the mean for the backbone atoms in the eight helices: 0.33 Å). Calcyclin exhibits a symmetric dimeric fold of two identical 90 amino acid subunits, characteristic of the S100 subfamily of EF-hand Ca2+-binding proteins. The structure reveals a readily identified pair of putative sites for binding of Zn2+. In order to accurately determine the structural features that differentiate the various S100 proteins, distance difference matrices and contact maps were calculated for the NMR structural ensembles of apo calcyclin and rat and bovine S100B. These data show that the most significant variations among the structures are in the positioning of helix III and in loops, the regions with least sequence similarity. Inter-helical angles and distance differences for the proteins show that the positioning of helix III of calcyclin is most similar to that of bovine S100B, but that the helix interfaces are more closely packed in calcyclin than in either S100B structure. Surprisingly large differences were found in the positioning of helix III in the two S100B structures, despite there being only four non-identical residues, suggesting that one or both of the S100B structures requires further refinement.     

Binding,folding and insertion of a β-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKV^dP^lPTKVKVKVK), an engineered AMP and anti-cancer β-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a “flip and dip” mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers.     

Mechanism of activation and inhibition of the HER4/ErbB4 kinase

HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.     

Long-residency hydration, cation binding, and dynamics of loop E/helix IV rRNA-L25 protein complex

Molecular dynamics simulations of RNA-protein complex between Escherichia coli loop E/helix IV (LE/HeIV) rRNA and L25 protein reveal a qualitative agreement between the experimental and simulated structures. The major groove of LE is a prominent rRNA cation-binding site. Divalent cations rigidify the LE major groove geometry whereas in the absence of divalent cations LE extensively interacts with monovalent cations via inner-shell binding. The HeIV region shows bistability of its major groove explaining the observed differences between x-ray and NMR structures. In agreement with the experiments, the simulations suggest that helix-alpha1 of L25 is the least stable part of the protein. Inclusion of Mg2+ cations into the simulations causes perturbation of basepairing at the LE/HeIV junction, which does not, however, affect the protein binding. The rRNA-protein complex is mediated by a number of highly specific hydration sites with long-residing water molecules and two of them are bound throughout the entire 24-ns simulation. Long-residing water molecules are seen also outside the RNA-protein contact areas with water-binding times substantially enhanced compared to simulations of free RNA. Long-residency hydration sites thus represent important elements of the three-dimensional structure of rRNA.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar Interactions in Configurational Transitions

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50–100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a “monkey-bar”. We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.